Project Summary Common variable immunodeficiency (CVID) is defined by severe antibody deficiency and is the most common symptomatic primary immunodeficiency. While immunoglobulin (Ig) replacement therapy limits frequency and severity of infections, chronic inflammation involving gastrointestinal (GI) tract, lungs, and other sites still occurs in half of CVID patients. The mechanisms and optimal therapies for CVID with these complications (CVIDc) remain elusive. Until we fill this gap in our knowledge, CVID morbidity and mortality persist at excessive levels. The genetic cause of CVID is typically unknown, but heterozygous mutations of NFKB1 are most common (~4%). NFKB1 encodes p105 which is processed into p50 to form NF-?B heterodimers that drive inflammatory gene expression. Alternatively, unprocessed p105 inhibits NF-?B-mediated transcription. Elevation of type 1 (Th1) cytokines and altered NF-?B signaling are defining, but unexplained, features of CVIDc. The most common mutations in CVIDc involve NFKB1, but little is known about their effects. Beyond CVID, NFKB1 polymorphisms associate with gastric cancer and NFKB1 knockout mice also develop gastritis. Along these lines, we found NFKB1 mutation in a CVIDc patient with severe gastritis that improved, for unclear reasons, with TNF blockade. Determining if NFKB1 mutation outside the p50 coding region increases type 1 cytokines and promotes gastritis will close key knowledge gaps in CVIDc and deepen understanding of a pathway implicated in many diseases. Our long-term goal is to devise therapy to precisely modulate NF-?B signaling to treat inflammatory disease. Our short-term goal is to understand how NFKB1 variant location and nature of NF-?B dysfunction promotes CVIDc. The central hypothesis is that mutation of NFKB1 sparing p50 increases type 1 cytokines and promotes gastritis by impairing p105-mediated regulation. We will test our hypothesis with two specific aims (1) Test the hypothesis that CVID NFKB1 variant location determines its effect upon NF-?B signaling and (2) Determine if p105 deficiency promotes TNF-driven neutrophilic gastritis using iPSC organoids. Results will identify pivotal features of NF-?B dysfunction underlying CVIDc and thereby provide insights into a broadly relevant pathway of inflammation. Our expertise in primary immunodeficiency and iPSC biology uniquely positions us to carry out this proposal. Novelty of our project includes the ability to study rare NFBK1 variants alongside samples from a biobank of 100+ CVID patients connected with clinical data, allowing us to elucidate aspects of NF-?B dysfunction pivotal to NFKB1 variants and the larger population of genetically undefined CVIDc with a similar cytokine and/or RNA sequencing profile. Thus, we will show how rare variants can empower research into complex CVID etiologies. Also, as the first application of iPSCs to CVID, this R21 research project will provide a foundational precedent that positions us to use this approach in future efforts to unravel mechanisms of CVID- associated genetic variants.